The project is concerned with the mechanism of action of thymidylate-synthetase (deoxyuridylate plus 5,10-methylenetetrahydrofolate yields thymidylate plus 7,8-dithydrofolate) from amethopterin-resistant Lactobacillus casei. The problems to be investigated include (a) the separation of enzyme molecules having two active sites from those with only one active site, (b) the use of phenylglyoxal in modification and localization of active site arginyl residues in thymidylate synthetase, (c) the use of bifunctional agents to crosslink nearby residues in the enzymic active site, (d) the characterization of binary complex and pseudo-ternary complex formation, and (c) chemical modification studies of arginyl, lysyl, and carboxylate side chains in dihydrofolate reductase.